DNA methyltransferase 3A continuous circulation colorimetric enzyme activity assayed with the product specification
(Chinese Version)
The main purpose
DNA methyltransferase 3A (DNMT3A) active enzyme continuous cycle colorimetric quantitative detection reagent is a kind of synthetic artificially synthesized methyl receptor substrate molecule and inhibitory complex, through DNA methyltransferase, adenosine isoform Hydrogen peroxide is formed in the cysteine ​​nucleosidase, adenine deaminase and xanthine oxidase reaction systems, and after using a chromogenic dye and a peroxidase, a change in the absorption peak of the quinone imine product is produced, that is, a colorimetric color is used. An authoritative and classical technique for determining enzyme activity in cell or tissue lysing samples. The technology has been carefully developed and successfully tested. It is suitable for detecting the activity of DNA methyltransferase 3A in various cell or tissue lysate suspension samples (animal, human, etc.). The product is strictly sterile, ready to use, simple in operation and stable in performance.
technical background
Methylation of biomolecules plays an important role in various biological systems, such as signaling, biosynthesis, protein repair, gene silencing, and chromatin regulation. Adding a methyl group to the 5' cytosine on the CpG dinucleotide to become 5'-methylcytosine is an important appearance of mammalian cells. Genetic modification, associated with embryonic development, tumorigenesis, and genetic disease. DNA methyltransferase (DNMT) catalyzes the maintenance and production of this methyl modification. There are three types of DNA methyltransferases: DNMT1, DNMT3a, and DNMT3b. DNMT1 acts to maintain methylation status, while DNMT3 is responsible for establishing new de novo methylation. Based on procainamide, a non-nucleotide inhibitor that specifically inhibits DNA methyltransferase 1 activity, and trichostatin A (TSA), an antifungal antibiotic, Specific inhibition of DNA methyltransferase 3B activity, participate in its reaction system, namely S-adenosylmethionine (SAM), also known as AdoMet, as the most commonly used methyl direct donor, The methyl group is transferred to the synthetic methyl acceptor substrate molecule poly[d(IC) d(IC) by DNA methyltransferase to form S-adenosylhomocysteine; AdoHcy), which is rapidly decomposed by adenosine homocysteine ​​nucleosidase into S-ribosylhomocysteine ​​and adenine, avoiding the negative feedback inhibition of the former. Methylation process. Adenine produces hypoxanthine from adenine deaminase, which is catalyzed by xanthine oxidase to form hydrogen peroxide, which is then reacted with p-hydroxybenzoic acid by the action of peroxidase ( P- hydroxybenzoic acid reacts with 4-aminoantipyrine to form a quinoneimine, and the activity of DNA methyltransferase 3A is quantitatively analyzed by a change in its absorption peak (wavelength at 510 nm). The DNA methyltransferase 3A reaction system is:
DNA methyltransferase 3A
Poly[d(IC) d(IC)] + AdoMet → methyl poly[d(IC) d(IC)] + S-adenosylhomocysteine
AdoHcy nucleosidase
S-adenosylhomocysteine ​​→ adenine + S-ribosylhomocysteine
Adenine deaminase
Adenine → hypoxanthine
Xanthine oxidase
Hypoxanthine + 2H2O + O2 → urate + 2H2O2
Peroxidase
2H2O2 + p -hydroxybenzoic acid + 4-aminoantipyrine → quinoneimine + 4H2O
product content
Buffer (Reagent A) 3 ml
Base Solution (Reagent B) 250 μl
Reaction solution (Reagent C) 250 μl
Negative Liquid (Reagent D) 250 μl
Obligate liquid (Reagent E) 250 μl
Product manual 1 copy
storage method
Store in a -20 ° C refrigerator; Reagent B to avoid light; effective to ensure June
User-supplied
100 microliter 1 cm light path cuvette or 96-well plate: for colorimetric containers
Spectrophotometer or microplate reader: for colorimetric analysis
Experimental procedure
- Preparation for measurement
- Turn on and set the spectrophotometer (temperature is 37 ° C): wavelength 510nm, interval 5 minutes, reading 3 times (15 minutes total), and set zero
- Was removed from the freezer -20 ℃ agent, placed in an ice melt trough; substrate solution (Reagent B) protected from light; buffer (Reagent A) was placed at room temperature equalization
- Background control
- Transfer 90 μl of buffer ( Reagent A ) to the new cuvette
- Add 10 μl of specific liquid ( Reagent E )
- Add 10 μl of substrate solution ( Reagent B )
- Add 10 μl of reaction solution ( Reagent C )
- Incubate for 3 minutes at 37 ° C
- Add 5 μl of negative solution ( Reagent D )
- Pour up and down several times, mix (limited to 3 seconds)
- Immediately put into the spectrophotometer test, this is the background air control (15 minutes reading - 0 minutes reading)
- Sample activity assay
- Transfer 90 μl of buffer ( Reagent A ) to the new cuvette
- Add 10 μl of specific liquid ( Reagent E )
- Add 10 μl of substrate solution ( Reagent B )
- Add 10 μl of reaction solution ( Reagent C )
- Incubate for 3 minutes at 37 ° C
- Add 5 μl of the sample to be tested (20 μg nucleoprotein) (Note: the sample should be clear )
- Pour up and down several times, mix (limited to 3 seconds)
- Immediately put into the spectrophotometer test, this is the sample reading (15 minutes reading - 0 minutes reading)
Fourth, calculate sample activity
[(Sample reading - background reading) X sample dilution factor X 0.125 (system capacity; ml)] ÷ [0.005 (sample capacity; ml) X 6.58 (mmol absorbance) X 15 (reaction time; minutes)] = unit / ÷ ÷ (sample protein concentration) mg / ml = unit / mg
Unit = micromolar S-adenosylmethionine / minute
- ELISA assay instrument
- Make the appropriate mark on the 96-well plate: background control and sample
- Transfer 90 μl of buffer ( Reagent A ) to a 96-well plate.
- Add 10 μl of specific liquid ( Reagent E )
- Add 10 μl of substrate solution ( Reagent B )
- Add 10 μl of reaction solution ( Reagent C )
- Incubate for 3 minutes at 37 ° C
- Add 5 μl of negative solution ( Reagent D ) or sample to be tested (20 μg of nucleoprotein) to the appropriate well (Note: the sample should be clear )
- Gently shake the 96-well plate
- Immediately put into the microplate reader test: 0 minute reading and 15 minute reading
- Activity calculation:
[(Sample reading - background reading) X sample dilution factor X 0.125 (ml, measured capacity)] ÷ [0.005 (sample capacity, ml) X 6.58 (mmol absorbance) X 0.6 (light path distance; cm) X 15 ( Reaction time; minutes)] = unit / ml ÷ (sample protein concentration) mg / ml = unit / mg
Unit = micromolar S-adenosylmethionine / minute
Precautions
- This product is 20 operations, including background control
- Wear gloves when handling
- Only 1 time for background determination during system operation
- Purified nuclear lysis suspension (20 μg nucleoprotein) is recommended as the sample to be tested
- Avoid using DTT, mercaptoethanol, TCEP, EDTA, etc. in the sample
- Samples must be clarified, it is vital
- Colorimetric determination within 3 seconds after loading
- The measured value changes from low to high; the measurement lasts for 15 minutes.
- After the colorimetric determination, the cuvette must be thoroughly cleaned.
- The sample is measured for 15 minutes and the reading is higher than 0 minutes.
- It is recommended that the sample protein concentration to be tested be 20 μg / 5 μl (the company provides Bradford Protein Concentration Quantitation Kit -30030.1)
- If the concentration of the sample to be tested is too high or too low, the sample concentration can be adjusted.
- The DNA methyltransferase 3A unit activity is defined as the amount of enzyme required to transfer 1 micromole of S-adenosylmethionine methyl group per minute as an active unit at 37 ° C, pH 8.0.
- The company provides a series of methylation detection reagent products
Quality Standard
- This product has been certified to be stable.
- This product has been identified and sensitive
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YT(Xi'an) Biochem Co., Ltd. , https://www.ytwholefood.com